Pentameric ligand-gated ion channels (LGICs) are activated by neurotransmitter binding to highly specialized, inter-subunit, extracellular binding pockets. In contrast, voltage-gated potassium (Kv) channels are activated by membrane depolarization, electromechanically communicated by the voltage sensor to the pore module. Kv channels composed of KCNQ2/3 heteromers generate the neuronal M-current, a ubiquitous and essential hyperpolarizing K+ current controlling excitability in mammalian CNS. This proposal is based on our recent discovery that KCNQ2/3 channels are directly activated by ?-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in vertebrate CNS, with sensitivity comparable to that of the most sensitive ?/?/? GABAA receptor LGICs. In contrast, the excitatory neurotransmitter glutamate, which is structurally related to GABA, has no effect on KCNQ2/3 activity. We have identified the KCNQ2/3 GABA binding site as KCNQ3-W265, and find the position is highly conserved in deuterostome clades, present in some Cnidarians, but absent in protostomes; it is also absent from cardiac-expressed KCNQ1. In addition, we have found that GABA analogs and metabolites exhibit similar structure-activity relationships (SARs) for KCNQ2/3 channel activation and anticonvulsant activity. The metabolites include ?-hydroxybutyrate (BHB), the primary ketone body produced in response to fasting or ketogenic diets, which protect against seizures. We find that BHB is a potent KCNQ2/3 activator and anticonvulsant, uncovering a molecular target for the therapeutic effects of ketosis. Our findings show that despite their wide structural disparity, GABA activates both principal classes of inhibitory ion channels in vertebrate neurons, creating a new paradigm for regulation of Kv channel gating and inhibitory neurotransmission. We propose three Specific Aims directed towards a fuller understanding of the mechanisms, breadth and scope underlying this novel signaling modality. In Aim 1 we will elucidate the molecular requirements for GABA and BHB regulation of KCNQ channels and when this capability evolved. In Aim 2 we will define the KCNQ binding sites of key GABA analogs and metabolites we recently discovered to also activate KCNQs, and leverage synergy between these compounds to develop optimized, potent anticonvulsants. In Aim 3, we will utilize newly CRISPR-Cas9 generated mice bearing germline mutations in the KCNQ3 & 5 GABA/BHB binding sites to determine the importance and KCNQ isoform-dependence of the anticonvulsant actions of BHB and the ketogenic diet. We will then use cellular electrophysiological analysis to quantify the age- and KCNQ-isoform dependence of GABA and BHB modulation of native M-current and neuronal excitability. The project will thoroughly define the fundamental aspects of a novel, unexpected form of inhibitory neuronal signaling, with the dual goals of understanding its role in brain physiology and harnessing the knowledge to help develop advanced, safer therapeutics for epilepsy and other neurological disorders.